Difference between revisions of "Part:BBa K2368006"

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__NOTOC__
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<h1>Introduction</h1>
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<partinfo>BBa_K2368006 short</partinfo>
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<h3>General</h3>
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<p>To measure the sweetness of sweeteners, we designed the detection circuit. This composite part is based on the <i>Saccharomyces cerevisiae</i> pheromone signaling pathway. It contains a yeast induced promoter <i>P<sub>fus</sub></i> , a  <i>mRFP </i> reporter, and a yeast terminator. In  <i>CEN.PK2-1C </i> (a mating type yeast) <i>P<sub>fus</sub></i> is activated by α pheromone, thereby initiating the expression of the reporter gene.</p>
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[[File:T_BIT-China_2017part_8.png|center|500px|默认文字]]
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<p style="text-align: center">Fig.1 The detection circuit</p>
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<p>We constructed <i>P<sub>fus</sub></i>-<i>mRFP-cyc1t-PRS42K</i> recombinant plasmid, and transformed it into <i>CEN.PK2-1C</i> (a type). </p>
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[[File:T_BIT-China_2017part_9.png|center|300px|默认文字]]
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<p style="text-align: center">Fig.2 The result of colony PCR</p>
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<p>Cells transformed with the signal reporter device were cultured to the exponential phase and treated with α pheromone.</p>
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<p>We added 5umol/L α pheromone into the transformed yeast. And after 4 hours, it was fluorescent.</p>
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<div style="margin-left: 270px"><ul>
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<li style="display: inline-block;"> [[File:T_BIT-China_2017part_10.png|center|center|220px|a]] </li>
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<li style="display: inline-block;"> [[File:T_BIT-China_2017part_11.png|center|center|220px|b]] </li>
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</ul></div>
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<p style="text-align: center">Fig.3 Images of yeast fluorescence. a: Initial fluorescence image; b: Fluorescence image after 4 hours.</p>
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<p>In order to characterize the function of detection circuit, we measured absorbance and fluorescence of the cells in the plate reader after treating them with varying concentrations of α pheromone.  </p>
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[[File:T_BIT-China_2017part_12.png|center|500px|默认文字]]
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<p style="text-align: center">Fig.4 <i>mRFP</i> intensity of <i>CEN.PK2-1C</i> induced by α factor</p>
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<p>As shown in this figure, adding higher concentration of α pheromone, the fluorescence intensity of the yeast increases. After 20 hours, α pheromone gradually failed, and the fluorescence intensity also decreased.</p>
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===Usage and Biology===
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<h2>Sequence and Features</h2>
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<partinfo>BBa_K2368006 SequenceAndFeatures

Latest revision as of 07:21, 24 October 2017


Introduction

Signal reporter device (Pfus-mRFP-CYC1t)

General

To measure the sweetness of sweeteners, we designed the detection circuit. This composite part is based on the Saccharomyces cerevisiae pheromone signaling pathway. It contains a yeast induced promoter Pfus , a mRFP reporter, and a yeast terminator. In CEN.PK2-1C (a mating type yeast) Pfus is activated by α pheromone, thereby initiating the expression of the reporter gene.

默认文字

Fig.1 The detection circuit

We constructed Pfus-mRFP-cyc1t-PRS42K recombinant plasmid, and transformed it into CEN.PK2-1C (a type).

默认文字

Fig.2 The result of colony PCR

Cells transformed with the signal reporter device were cultured to the exponential phase and treated with α pheromone.

We added 5umol/L α pheromone into the transformed yeast. And after 4 hours, it was fluorescent.

  • a
  • b

Fig.3 Images of yeast fluorescence. a: Initial fluorescence image; b: Fluorescence image after 4 hours.

In order to characterize the function of detection circuit, we measured absorbance and fluorescence of the cells in the plate reader after treating them with varying concentrations of α pheromone.

默认文字

Fig.4 mRFP intensity of CEN.PK2-1C induced by α factor

As shown in this figure, adding higher concentration of α pheromone, the fluorescence intensity of the yeast increases. After 20 hours, α pheromone gradually failed, and the fluorescence intensity also decreased.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 993
    Illegal AgeI site found at 1105
  • 1000
    COMPATIBLE WITH RFC[1000]